Molecular cloning and enzymological characterization of pyridoxal 5′-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473

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• 作者：Tsubasa Washio ; Shiro Kato ; Tadao Oikawa
• 关键词：Thermococcus litoralis ; Aspartate racemase ; d ; Aspartic acid ; Hyperthermophilic archaeon ; Molecular cloning
• 刊名：Extremophiles
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：20
• 期：5
• 页码：711-721
• 全文大小：752 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Biotechnology
  Ecology
  
• 出版者：Springer Japan
• ISSN：1433-4909
• 卷排序：20

文摘

We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5′-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on l-cysteic acid and l-cysteine sulfinic acid in addition to d- and l-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. d-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells.